Gene encoding a polypeptide having nitrile hydratase activity, a transformant containing the gene and a process for the production of amides using the transformant

ABSTRACT

The present invention relates to a gene derived from Pseudomonas chlororaphis B23 strain which encodes a polypeptide having nitrile hydratase activity being capable of hydrating nitriles to amides. The invention also relates to a recombinant DNA containing the gene, and a transformant transformed with the recombinant DNA. The present invention further relates to a method of producing nitrile hydratase using the transformant and of amides using nitrile hydratase.

This is a continuation of application Ser. No. 07/694,746, filed May 2,1991, abandoned.

FIELD OF THE INVENTION

The present invention relates to a gene which is derived fromPseudomonas chlororaphis B23 and which encodes a polypeptide havingnitrile hydratase activity to hydrate nitriles to amides. The inventionalso relates to a recombinant DNA containing the gene, and atransformant transformed with the recombinant DNA. The present inventionfurther relates to a method of the production of nitrile hydratase usingthe transformant and of amides using nitrile hydratase.

PRIOR ART

Nitrile hydratase or nitrilase is known as an enzyme that hydratesnitriles to amides. Microorganisms that produce nitrile hydrataseinclude those belonging to the genus Bacillus, the genus Bacteridium,the genus Micrococcus and the genus Brevibacterium (See,JP-B-62-21517/1989, U.S. Pat. No. 4,001,081), the genus Corynebacteriumand the genus Nocardia (See, JP-B-56-17918/1989, U.S. Pat. No.4,248,968), the genus Pseudomonas (See, JP-B-59-37951/1984, U.S. Pat.No. 4,637,982), the genus Rhodococcus, the genus Arthrobacter and thegenus Microbacterium (See, JP-A-61-162193/1986, EP-A-0188316), andRhodococcus rhodochrous (See, JP-A-2-470/1990, EP-A-0307926).

Nitrile hydratase has been used to hydrate nitriles to amides. In theinvention, microorganisms are engineered to contain multiple copies of arecombinant DNA encoding nitrile hydratase according to a recombinantDNA technology. The recombinant produces a remarkably high level ofnitrile hydratase compared with conventional methods.

The present inventors previously disclosed a gene derived fromRhodococcus sp. N-774 (FERM BP-1936) which also encodes a polypeptidehaving nitrile hydratase activity (JP-A-2-119778/1988).

In contrast, the present inventors utilized a gene derived fromPseudomonas chlororaphis B23 described in said U.S. Pat. No. 4,637,982for the production of nitrile hydratase. We isolated the gene encodingnitrile hydratase, inserted the gene into a suitable plasmid vector andtransformed an appropriate host with the recombinant plasmid, and thussuccessfully obtained the transformant producing nitrile hydratase.

SUMMARY OF THE INVENTION

The present invention relates to

(1) a gene encoding a polypeptide which has nitrile hydratase activityand which comprises α-subunit of the following amino acid sequence asdefined in the Sequence Listing by SEQ ID: No. 1, and β-subunit of thefollowing amino acid sequence as defined in the Sequence Listing by SEQID: No. 2.

(2) a gene as described in (1) encoding α- and β-subunits, comprisingα-subunit-encoding sequence as defined in the Sequence Listing by SEQID: No. 3, and β-subunit-encoding sequence as defined in the SequenceListing by SEQ ID: No. 4.

(3) a recombinant DNA Comprising a vector containing the gene asdescribed in (1) or (2);

(4) a transformant transformed with the recombinant DNA as described in(3);

(5) a method for the production of nitrile hydratase which comprisesculturing the transformant as described in (4) and recovering nitrilehydratase from the culture;

(6) a method for the production of amides which comprises hydratingnitriles using nitrile hydratase as described in (6) to form amides; and

(7) a method for the production of amides which comprises culturing thetransformant as described in (4), and hydrating nitriles using theresultant culture isolated bacterial cells, treated matter thereof, or afixed material of them, to form amides.

The present invention is described in detail as follows.

(1) Isolation and Purification of Nitrile Hydratase and Partial AminoAcid Sequencing of Nitrile Hydratase Nitrile hydratase is isolated andpurified from Pseudomonas chlororaphis B23 and separated into α and βsubunits using HPLC. N-Terminal amino acid sequence of the subunits isdetermined and shown in the Sequence Listing by SEQ ID: No. 5 and No. 6,respectively.

(2) Preparation of a DNA Probe for a Nitrile Hydratase Gene

A DNA probe is prepared from JM105/pYUK121 (FERM BP-1937) as describedin JP-A-2-119778/1990 due to the high degree of homology in the aminoacid sequence between the nitrile hydratase β subunit of Rhodococcus sp.N-774 described in said Japanese Patent official gazette and that ofPseudomonas chlororaphis B23. Plasmid pYUK121 containing nitrilehydratase gene derived from Rhodococcus sp. N-774 is prepared from aJM105/pYUK121 culture. pYUK121 DNA is digested with SphI and SalI. TheSphI-SalI fragment contains the nitrile hydratase gene (shown in theSequence Listing as defined by SEQ ID: No. 7) of Rhodococcus sp. N-774.The DNA fragment is radiolabeled to give a probe.

(3) Detection of a DNA Segment Containing a Nitrile Hydratase Gene fromthe Chromosome of Pseudomonas chlororaphis B23

Chromosomal DNA is prepared from a Pseudomonas chlororaphis B23 culture.Chromosomal DNA is digested with restriction enzymes and hybridized tothe probe described in (2), according to a Southern hybridization method[Southern, E. M., J. Mol. Biol. 98, 503 (1975)]. Two DNA fragments ofdifferent length are screened.

(4) Construction of a Recombinant Plasmid

A recombinant plasmid is constructed by inserting the chromosomal DNAfragment as prepared in (3) into a plasmid vector.

(5) Transformation and Screening of the Transformant Containing theRecombinant Plasmid

Transformants are prepared using the recombinant plasmid as described in(4). The transformant containing the recombinant plasmid is selectedusing the probe as described in (2) according to a colony hybridizationmethod [R. Bruce Wallace et. al., Nuc. Aci. Res. 9, 879 (1981)].Additionally, the presence of the nitrile hydratase gene in therecombinant plasmid is confirmed using a Southern hybridization method.The plasmids thus selected are designated as pPCN1 and pPCN3.

(6) Isolation and Purification of Plasmid DNA and Construction of theRestriction Map

Plasmid DNAs of pPCN1 and pPCN3 as described in (5) are isolated andpurified. The restriction maps of the DNAs are constructed (FIG. 1) todetermine the region containing nitrile hydratase gene.

(7) DNA Sequencing

The extra segment of the inserted DNA fragment in pPCN1 and pPCN3 isexcised using an appropriate restriction enzyme. The inserted DNAfragment is then used for sequencing. The nucleotide sequence of the DNAfragment. (see, the Sequence Listing as defined by SEQ ID: No. 8)reveals that it contains the sequence deduced from the amino acidsequence as described in (1).

(8) Insertion of the DNA Fragment into an Expression Vector andTransformation

The DNA fragment is cleaved from pPCN1 and pPCN3 usingappropriate-restriction enzymes. These two fragments are ligated andinserted into an expression vector pUC19. The construct is used fortransformation of E. coli JM105 (Amersham), and the transformant isdesignated as JM105/pPCN4.

(9) Production of Nitrile Hydratase Using the Transformant andConversion of Nitriles to Amides

The transformant as described in (8) is cultured. The bacterial cellsare mixed with nitriles, a substrate of nitrile hydratase, and amidesare produced.

Pseudomonas chlororaphis B23 was deposited with Fermentation ResearchInstitute, Agency of Industrial Science and Technology presently calledNational Institute of Bioscience and Human Technology, the Agency ofIndustrial Science and Technology, 1-3 Higashi 1-chome, Tsukuba-shi,Ibaraki-ken, 305, Japan. and was assigned the accession number FERMBP-187. A transformant JM105/pPCN4 was deposited with the same on Feb.27, 1990 and assigned the accession number FERM BP-2779.

Any vector including a plasmid vector (e.g., pAT153, pMP9, pHC624, pKC7,etc.), a phage vector (e.g., λgt11 (Toyobo), Charon 4A (Amersham), etc.may be used for the present invention. Enzymes which may be used includeSphI, SalI, SacI, BamHI, EcoRI, PstI and the like, which arecommercially available (Takara Shuzo). Various hosts may be used fortransformation including but not limited to E. coli JM105 and TG1.Culture media for the transformant are those ordinarily used in the art.

Conversion of nitriles to amides is carried out using nitrile hydratase,crude nitrile hydratase, the culture of the transformant, the isolatedbacterial cells or treated matter thereof, and the like, prepared fromthe culture of the transformant.

Suitable nitriles include those having 2-4 carbon atoms such asacetonitrile, propionitrile, acrylonitrile, methacrylonitrile,n-butyronitrile and isobutyronitrile, and acrylonitrile is preferred.

DESCRIPTION OF THE FIGURES

FIG. 1 shows restriction maps of recombinant plasmids, pPCN1, pPCN3 andpPCN4.

THE EFFECT OF THE INVENTION

The present invention discloses the amino acid sequence and nucleotidesequence of the α- and β-subunits of nitrile hydratase derived fromPseudomonas chlororaphis B23. The gene encoding nitrile hydratase isinserted into an expression vector and the recombinant vector is usedfor transformation. The transformant contains multiple copies of thegene and produces a higher level of nitrile hydratase compared withconventionally used microorganisms.

The present invention will be described in detail in the followingExample which should not be construed to limit the invention.

The following abbreviations are used in Example.

TE: Tris-HCl (10 mM; pH 7.8), EDTA (1 mM, pH 8.0)

TNE: Tris-HCl (50 mM; pH 8.0), EDTA (1 mM, pH 8.0), NaCl (50 mM)

STE: Tris-HCl (50 mM; pH 8.0), EDTA (5 mM, pH 8.0), Sucrose (35 mM)

2×YT medium: 1.6% Trypton; 1.0% Yeast extract, 0.5% NaCl

EXAMPLE

(1) Isolation and Purification of Nitrile Hydratase and the Amino AcidSequencing of a Portion of Nitrile Hydratase

Pseudomonas chlororaphis B23 was cultured in a medium (10 g/l ofsucrose, 4 g/l of methacrylamide, 0.5 g/l of KH₂ PO₄, 0.5 g/l of K₂HPO₄, 0.5 g/l of MgSO₄.7H₂ O, 0.01 g/l of FeSO₄.7H₂ O, pH 7.0) at 25° C.for 28 hours. The bacterial cells were harvested. 100 g of the bacterialcells was disrupted and fractionated with ammonium sulfate. The samplewas dialyzed and the dialysate was centrifuged. The supernatant wasremoved and subjected to DEAE-Sephacel chromatography, Octyl-SepharoseCL-4B chromatography, Phenyl-Sepharose CL-4B chromatography and SephadexG-150 chromatography. Active fractions were collected and dialyzed. Thedialysate containing the enzyme was subjected to a high performanceliquid chromatography using a reversed phase column (Senshu PakVP-304-1251, Senshu Kagaku), and to subunits (α and β) were obtained.The N-terminal amino acid sequences of α- and β-subunits were determinedusing an amino acid sequencer (Applied Biosystems model 470A).N-terminal amino acid sequences of α- and β-subunits are shown in theSequence Listing as defined by SEQ ID: No. 5 and SEQ ID: No. 6respectively.

(2) Preparation of a DNA Probe for Nitrile Hydratase Gene

E. coli JM105 containing pYUK121 (FERM BP-1937) was cultured in 100 mlof 2×YT medium containing 50 μg/ml of ampicillin at 30° C. overnight (12hours). The bacterial cells were harvested and TNE was added to thecells. The cell suspension was then centrifuged. 8 ml of STE and 10 mgof lysozyme were added to the pellet. The mixture was incubated at 0° C.for five minutes followed by the addition of 4 ml of 0.25M EDTA. 2 ml of10% SDS and 5 ml of 5M NaCl were then added to the mixture at roomtemperature. The resultant mixture was left standing at 0°-4° C. forthree hours and then ultracentrifuged. 1/2 volume of 30% PEG 6000 wasadded to the supernatant. The mixture was allowed to stand at 0°-4° C.overnight (12 hours) and centrifuged. TNE was added to the pellet tobring the volume to 7.5 ml and CsCl was then added to the suspension.The mixture was centrifuged to remove proteins. Then, 300-500 mg/ml ofethidium bromide was added to the supernatant. The mixture wastransferred to a centrifuge tube. The tube was heat-sealed and thenultracentrifuged. cccDNA. was extracted using a peristaltic pump. A bitmore than equal volume of isopropyl alcohol saturated with water wasadded to the extract to remove ethidium bromide. The sample was dialyzedagainst TE. About 3 ml of purified pYUK121 was obtained.

pYUK121 DNA was digested with SphI and SalI, resulting in a 2.07 kb DNAfragment containing a nitrile hydratase gene derived from Rhodococcussp. N-774. The fragment was radiolabeled with ³² P to produce a probe.The nucleotide sequence of the probe is shown in the Sequence Listing asdefined by SEQ ID: No. 7.

(3) Preparation of a DNA Fragment Containing a Nitrile Hydratase Gene ofChromosome

Pseudomonas chlororaphis B23 was cultured in 100 ml of the medium asdescribed in (1). The bacterial cells were harvested and washed withTNE. The pellet was then suspended in 10 ml of TE. 4 ml of 0.25M EDTA,10-20 mg of lysozyme, 10-20 mg of achromoprotease and 10 ml of 10% SDSwere added to the suspension. The mixture was incubated at 37° C. forthree hours. 15 ml of phenol was added to the mixture. The mixture wasincubated at room temperature for 15 minutes and then centrifuged. To 15ml of the upper layer, 0.7 ml of 2.5M sodium acetate and diethyl etherwere added and the mixture was centrifuged. The upper layer wasdiscarded. Two volumes of ethanol were added to the bottom layer and DNAwas removed with a glass rod. DNA was rinsed with TE:ethanol 2:8, 1:9,and 0:10 (v/v) for five minutes each. DNA was then resuspended in 2-4 mlof TE (37° C.). 10 μl of a mixture of RNase A and T₁ was added to thesuspension and the mixture was incubated at 30° C. An equal volume ofphenol was added to the mixture. The mixture was then centrifuged. Aftercentrifugation, 2-4 ml of the upper layer was removed. More than equalamount of ether was added to the removed portion. The mixture wascentrifuged. After centrifugation, the upper layer was discarded. Thebottom layer was dialyzed overnight against 2 l of TE containing a smallamount of chloroform and further dialyzed against fresh TE for 3-4hours. 4ml of crude chromosomal DNA was obtained. Enzymatic digestion ofchromosomal DNA was carried out as follows:

(a) 2 μl of SacI+3 μl of reaction buffer (10×)+15 μl of chromosomalDNA+10 μl of TE

(b) 2 μl of BamHI+2 μl of SalI+3 μl of reaction buffer (10×)+15 μl ofchromosomal DNA+8 μl of TE

The mixtures were incubated at 37° C. for an hour and electrophoresed onan agarose gel at 60 V for three hours. The Southern hybridization ofchromosomal DNA was carried out using the probe as described in (2).Fragments of about 4.6 kb and 4.7 kb were found to show a stronghybridization.

15 μl of chromosomal DNA was digested with SacI, and BamI and SalI andelectrophoresed on an agarose gel as described above. 4.6 kb and 4.7 kbDNA fragments were cut out from the gel and placed in three volumes of8M NaClO₄. The solution was dotted on a GF/C (Whatman) filter paper (6mm in diameter). Ten drops (≃100 μl) of TE containing 6M NaClO₄ and thenten drops (≃100 μl) of 95% ethanol were added to the filter paper. Thepaper was air-dried and placed in 0.5 ml Eppendorf tube. 40 μl of TE wasadded to the tube and the tube was incubated at 37° C. for 30 minutes.The tube was then centrifuged. About 40 μl of the supernatant wasobtained which contained 4.6 kb and 4.7 kb DNA fragments containing anitrile hydratase gene of chromosomal DNA.

(4) Insertion of the Chromosomal DNA Fragment into a Vector

(a) 4.6 kb DNA Fragment

2 μl of SacI, 3 μl of reaction buffer (10×) and 10 μl of TE were addedto 10 μl of pUC18 DNA. The mixture was incubated at 37° C. for an hour.2 μl of 0.25M EDTA was added to the mixture to stop the reaction. Then 7μl of 1M Tris-HCl (pH 9) and 3 μl of BAP (bacterial alkalinephosphatase) were added to the mixture. The mixture was incubated at 65°C. for an hour. TE was then added to the mixture to bring the volume to100 μl. The mixture was extracted 3×with an equal volume of phenol. Anequal volume of ether was added to the extract. The bottom layer wasremoved and 10 μl of 3M sodium acetate and 250 μl of ethanol were addedto the bottom layer. The mixture was incubated at -80° C. for 30minutes, centrifuged, dried, and resuspended in TE.

5 μl of pUC18 DNA thus obtained and 40 μl of the 4.6 kb DNA fragment asdescribed in (3) were mixed. 6 μl of ligation buffer, 6 μl of ATP (6mg/ml) and 3 μl of T4 DNA ligase were added to the mixture. The mixturewas incubated at 4° C. overnight (12 hours) to produce the recombinantplasmid containing the 4.6 kb DNA fragment in the SacI site of pUCI8.

(b) 4.7 kb DNA Fragment

pUCl8 DNA was digested with BamHI and SalI. The 4.7 kb DNA fragment wasinserted into the BamHI-SalI site of pUCl8 in a similar manner asdescribed in (4)-(a). The recombinant plasmid containing the 4.7 kb DNAfragment in the BamHI-SalI site of pUCI8 was obtained.

(5) Transformation and Screening of Transformants

E. coli JM105 (Amersham) was inoculated into 10 ml of 2×YT medium andincubated at 37° C. for 12 hours. After incubation, the culture wasadded to a fresh 2×YT medium to a final concentration of 1%, and themixture was incubated at 37° C. for two hours. The culture wascentrifuged and the pellet was suspended in 5 ml of cold 50 mM CaCl₂.The suspension was placed at 0° C. for 40 minutes and then centrifuged.0.25 ml of cold 50 mM CaCl₂ and 60 μl each of the recombinant plasmidsas prepared in (4)(a) and (b) were added to the pellet in a separatetube. The mixture was incubated on ice for 40 minutes, heat-shocked at42° C. for two minutes, placed on ice for five minutes, and added to 10ml of 2×YT medium. The mixture was incubated at 37° C. for 90 minuteswith shaking, then centrifuged. The pellet was suspended in 1 ml of 2×YTmedium, and 10 μl of the suspension was plated on a 2×YT agar platecontaining 50 μg/ml of ampicillin. The plate was incubated at 37° C. Thecolony grown on the plate was selected by the colony hybridizationmethod: The colony was transferred to a nitrocellulose filter and lysed.The DNA fixed on the filter was hybridized to the probe as described in(2). The filter was autoradiographed and a recombinant colony wasselected. Additionally, the presence of a nitrile hydratase gene in thetransformant was confirmed according to the Southern hybridizationmethod.

(6) Isolation and Purification of Recombinant Plasmid and Constructionof the Restriction Map of the Inserted DNA Fragments

The transformant selected as described in (5) was grown in 100 ml of a2×YT medium containing 50 μg/ml of ampicillin at 37° C. overnight (12hours). The bacterial cells were harvested and TNE was added to thecells. The cells were collected again by centrifugation, and 8 ml of STEand 10 mg of lysozyme were added to the cells. The mixture was incubatedat 0° C. for five minutes. 4 ml of 0.25M EDTA, 2 ml of 10% SDS (at roomtemperature) and 5 ml of 5M NaCl were added to the mixture. The mixturewas incubated at 0°-4° C. for three hours, and ultracentrifuged. 1/2volume of 30% PEG 6000 was added to the supernatant. The mixture wasincubated at 0°-4° C. overnight (12 hours) and centrifuged again. TNEwas added to the pellet to bring the volume up to 7.5 ml. CsCl was addedto the mixture. The mixture was centrifuged to remove proteins. Then,300-500 mg/ml of ethidium bromide was added to the supernatant and themixture was transferred to a centrifuge tube. The tube was heat-sealedand ultracentrifuged. cccDNA was removed using a peristaltic pump. A bitmore than equal volume of isopropyl alcohol saturated with water wasadded to cccDNA to remove ethidium bromide. The DNA sample was dialyzedagainst TE, resulting in about 3 ml of recombinant DNA. The recombinantplasmid containing a 4.6 kb DNA fragment was designated as pPCN1 (Therecombinant plasmid containing a 4.7 kb DNA fragment was designated aspPCN3).

These plasmid DNAs were digested with EcoRI, BamHI, PstI, SacI and SalI.The restriction maps were constructed and are shown in FIG. 1.

(7) DNA Sequencing

The location of a nitrile hydratase gene in the DNA fragments of pPCN1and pPCN3 was determined according to the restriction map constructedand to the Southern hybridization method. Based on the results, the2.456 kb BamHI-HincII DNA fragment was sequenced by the Sanger method[Sanger, F., Science 214:1205-1210 (1981)] using M13 phage vector. The2.456 kb DNA fragment from Pseudomonas chlororaphis B23 is shown in theSequence Listing as defined by SEQ ID: No. 8.

All the nucleotide sequence deduced from the amino acid sequence asdetermined in (1) was found in the sequence as determined above. Thesequence analysis also revealed that the DNA fragment contained thesequence coding for the α- and β-subunits.

(8) Insertion of the BamHI-HincII DNA Fragment into an Expression Vectorand Transformation

2.2 kb SphI-BamHI fragment of pPCN1 and 4.7 kb BamHI-SalI fragment ofpPCN3 were cleaved and both fragments were ligated (FIG. 1). The ligatedfragment was inserted into the SphI-SalI site of an expression vectorpUC19 and the construct was designated as pPCN4.

E. coli JM105 was transformed with pPCN4 in a similar manner as in (5)and the transformant was designated as JM105/pPCN4 (FERM BP-2779).

(9) Production of Nitrile Hydratase Using the Transformant andConversion of Nitriles to Amides Using Nitrile Hydratase

JM105/pPCN4 was inoculated into 10 ml of 2×YT medium containing 50 μg/mlof ampicillin and incubated at 26.5° C. overnight (12 hours). 100 μl ofthe resultant culture was added to 10 ml of 2×YT medium (50 μg/ml ofampicillin, 50 mg/l of FeSO₄.7H₂ O, 10 mg/l of MgSO₄.7H₂ O, 1 mg/l ofpyrroloquinolinequinone). The mixture was incubated at 26.5° C. for fivehours. IPTG was added to the mixture to a final concentration of 1 mM.The mixture was incubated at 26.5° C. for 10 hours. After harvesting thecells, the cells were suspended in 5 ml of 1/20M phosphate buffer (pH7.7). 10 μl of the substrate solution (1M acrylonitrile) was added to0.1 ml of the suspension. The mixture was incubated at 20° C. for 20minutes. As a control, the mixture obtained by the same procedure asdescribed above but from E. coli JM105 was used. The reaction mixturewas tested for the presence of acrylamide (the product of the enzymaticreaction) and acrylonitrile using HPLC. Acrylamide but no acrylonitrilewas found in the reaction mixture of JM105/pPCN4, whereas acrylonitrilebut no acrylamide was found in the reaction mixture of JM105. A numberof references are cited herein, the disclosures of which, in theirentireties are incorproated herein by reference.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 8                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 200 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Pseudomonas chlororaphis                                        (B) STRAIN: B23 (FERM BP-187)                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       MetSerThrSerIleSerThrThrAlaThrProSerThrProGlyGlu                              151015                                                                        ArgAlaTrpAlaLeuPheGlnValLeuLysSerLysGluLeuIlePro                              202530                                                                        GluGlyTyrValGluGlnLeuThrGlnLeuMetAlaHisAspTrpSer                              354045                                                                        ProGluAsnGlyAlaArgValValAlaLysAlaTrpValAspProGln                              505560                                                                        PheArgAlaLeuLeuLeuLysAspGlyThrAlaAlaCysAlaGlnPhe                              65707580                                                                      GlyTyrThrGlyProGlnGlyGluTyrIleValAlaLeuGluAspThr                              859095                                                                        ProGlyValLysAsnValIleValCysSerLeuCysSerCysThrAsn                              100105110                                                                     TrpProValLeuGlyLeuProProGluTrpTyrLysGlyPheGluPhe                              115120125                                                                     ArgAlaArgLeuValArgGluGlyArgThrValLeuArgGluLeuGly                              130135140                                                                     ThrGluLeuProSerAspThrValIleLysValTrpAspThrSerAla                              145150155160                                                                  GluSerArgTyrLeuValLeuProGlnArgProGluGlySerGluHis                              165170175                                                                     MetSerGluGluGlnLeuGlnGlnLeuValThrLysAspValLeuIle                              180185190                                                                     GlyValAlaLeuProArgValGly                                                      195200                                                                        (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 220 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Pseudomonas chlororaphis                                        (B) STRAIN: B23 (FERM BP-187)                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetAspGlyPheHisAspLeuGlyGlyPheGlnGlyPheGlyLysVal                              151015                                                                        ProHisThrIleAsnSerLeuSerTyrLysGlnValPheLysGlnAsp                              202530                                                                        TrpGluHisLeuAlaTyrSerLeuMetPheValGlyValAspGlnLeu                              354045                                                                        LysLysPheSerValAspGluValArgHisAlaValGluArgLeuAsp                              505560                                                                        ValArgGlnHisValGlyThrGlnTyrTyrGluArgTyrIleIleAla                              65707580                                                                      ThrAlaThrLeuLeuValGluThrGlyValIleThrGlnAlaGluLeu                              859095                                                                        AspGlnAlaLeuGlySerHisPheLysLeuAlaAsnProAlaHisAla                              100105110                                                                     ThrGlyArgProAlaIleThrGlyArgProProPheGluValGlyAsp                              115120125                                                                     ArgValValValArgAspGluTyrValAlaGlyHisIleArgMetPro                              130135140                                                                     AlaTyrValArgGlyLysGluGlyValValLeuHisArgThrSerGlu                              145150155160                                                                  GlnTrpProPheProAspAlaIleGlyHisGlyAspLeuSerAlaAla                              165170175                                                                     HisGlnProThrTyrHisValGluPheArgValLysAspLeuTrpGly                              180185190                                                                     AspAlaAlaAspAspGlyTyrValValValAspLeuPheGluSerTyr                              195200205                                                                     LeuAspLysAlaProGlyAlaGlnAlaValAsnAla                                          210215220                                                                     (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 600 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Pseudomonas chlororaphis                                        (B) STRAIN: B23 (FERM BP-187)                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       ATGAGTACATCTATTTCCACGACTGCGACACCTTCGACACCCGGCGAGAGGGCATGGGCC60                TTGTTTCAAGTGCTCAAGAGCAAGGAACTCATCCCAGAGGGCTATGTCGAGCAGCTCACT120               CAATTGATGGCCCATGACTGGAGCCCGGAGAACGGCGCTCGCGTGGTCGCCAAGGCATGG180               GTCGATCCGCAGTTCCGGGCGCTGCTGCTCAAGGACGGAACAGCCGCTTGCGCGCAGTTC240               GGCTACACCGGCCCACAAGGCGAATACATCGTCGCCCTGGAAGATACACCGGGGGTGAAG300               AACGTCATCGTCTGCAGCCTGTGCTCCTGCACCAACTGGCCGGTCCTCGGCCTGCCGCCC360               GAGTGGTACAAGGGCTTTGAGTTTCGTGCGCGCCTGGTCCGGGAGGGGCGCACCGTACTG420               CGCGAGCTGGGGACGGAGTTGCCGAGCGACACGGTCATCAAAGTCTGGGATACCAGCGCC480               GAAAGCCGTTACCTGGTGTTGCCGCAAAGGCCTGAAGGCTCTGAGCACATGAGTGAAGAA540               CAGCTTCAACAGCTGGTGACCAAAGACGTGCTGATTGGCGTCGCCCTGCCACGCGTTGGC600               (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 660 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Pseudomonas chlororaphis                                        (B) STRAIN: B23 (FERM BP-187)                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       ATGGATGGCTTTCACGATCTCGGCGGTTTCCAAGGCTTTGGCAAAGTGCCGCACACCATC60                AACAGCCTCAGCTACAAACAGGTTTTCAAGCAGGACTGGGAACACCTGGCCTATAGCTTG120               ATGTTTGTCGGCGTTGACCAATTGAAAAAGTTCAGCGTGGACGAAGTGCGTCATGCCGTC180               GAACGCCTGGACGTTCGCCAGCATGTCGGCACCCAGTACTACGAACGCTACATCATCGCG240               ACCGCCACGCTGCTGGTGGAAACGGGCGTTATCACCCAGGCGGAGCTCGATCAGGCATTG300               GGTTCCCACTTCAAGCTGGCGAACCCCGCCCATGCGACAGGTCGCCCGGCGATCACCGGC360               AGGCCGCCCTTCGAAGTGGGCGATCGGGTTGTGGTTCGAGACGAATATGTGGCGGGGCAT420               ATCCGCATGCCGGCCTACGTGCGCGGTAAGGAAGGCGTGGTCCTGCACCGCACCTCAGAG480               CAGTGGCCCTTCCCCGACGCCATTGGCCACGGCGACTTGAGCGCAGCCCATCAGCCTACC540               TACCACGTCGAGTTTCGCGTGAAAGATCTATGGGGTGACGCGGCAGATGACGGTTACGTC600               GTGGTCGATCTTTTCGAAAGCTACTTGGATAAGGCCCCCGGTGCCCAAGCGGTGAACGCA660               (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Pseudomonas chlororaphis                                        (B) STRAIN: B23 (FERM BP-187)                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       SerThrSerIleSerThrThrAlaThrProSerThrProGlyGluArg                              151015                                                                        AlaTrpAlaLeuPheGlnValLeuLys                                                   2025                                                                          (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Pseudomonas chlororaphis                                        (B) STRAIN: B23 (FERM BP-187)                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       MetAspGlyPheHisAspLeuGlyGlyPheGlnGlyPheGlyLysVal                              151015                                                                        ProHisThrIleAsnSerLeu                                                         20                                                                            (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2070 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Rhodococcus                                                     (B) STRAIN: sp. N-774 (FERM BP- 1936)                                         (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 675..1295                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1325..1960                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       GCATGCTTTCCACATCTGGAACGTGATCGCCACGGACGGTGGTGCCTACCAGATGTTGGA60                CGGCAACGGATACGGCATGAACGCCGAAGGTTTGTACGATCCGGAACTGATGGCACACTT120               TGCTTCTCGACGCATTCAGCACGCCGACGCTCTGTCCGAAACCGTCAAACTGGTGGCCCT180               GACCGGCCACCACGGCATCACCACCCTCGGCGGCGCGAGCTACGGCAAAGCCCGGAACCT240               CGTACCGCTTGCCCGCGCCGCCTACGACACTGCCTTGAGACAATTCGACGTCCTGGTGAT300               GCCAACGCTGCCCTACGTCGCATCCGAATTGCCGGCGAAGGACGTAGATCGTGCAACCTT360               CATCACCAAGGCTCTCGGGATGATCGCCAACACGGCACCATTCGACGTGACCGGACATCC420               GTCCCTGTCCGTTCCGGCCGGCCTGGTGAACGGGGTTCCGGTCGGAATGATGATCACCGG480               CAGACACTTCGACGATGCGACAGTCCTTCGTGTCGGACGCGCATTCGAAAAGCTTCGCGG540               CCGGTTTCCGACGCCGGCCGAACGCGCCTCCAACTCTGCACCACAACTCAGCCCCGCCTA600               GTCCTGACGCACTGTCAGACAACAAATTCCACCGATTCACACATGATCAGCCCACATAAG660               AAAAGGTGAACCAGATGTCAGTAACGATCGACCACACAACGGAGAACGCC710                         MetSerValThrIleAspHisThrThrGluAsnAla                                          1510                                                                          GCACCGGCCCAGGCGGCGGTCTCCGACCGGGCGTGGGCACTGTTCCGC758                           AlaProAlaGlnAlaAlaValSerAspArgAlaTrpAlaLeuPheArg                              152025                                                                        GCACTCGACGGTAAGGGATTGGTACCCGACGGTTACGTCGAGGGATGG806                           AlaLeuAspGlyLysGlyLeuValProAspGlyTyrValGluGlyTrp                              303540                                                                        AAGAAGACCTCCGAGGAGGACTTCAGTCCAAGGCGCGGAGCGGAATTG854                           LysLysThrSerGluGluAspPheSerProArgArgGlyAlaGluLeu                              45505560                                                                      GTAGCGCGCGCATGGACCGACCCCGAGTTCCGGCAGCTGCTTCTCACC902                           ValAlaArgAlaTrpThrAspProGluPheArgGlnLeuLeuLeuThr                              657075                                                                        GACGGTACCGCCGCAGTTGCCCAGTACGGATACCTGGGCCCCCAGGCG950                           AspGlyThrAlaAlaValAlaGlnTyrGlyTyrLeuGlyProGlnAla                              808590                                                                        GCCTACATCGTGGCAGTCGAAGACACCCCGACACTCAAGAACGTGATC998                           AlaTyrIleValAlaValGluAspThrProThrLeuLysAsnValIle                              95100105                                                                      GTGTGCTCGCTGTGTTCATGCACCGCGTGGCCCATCCTCGGTCTGCCA1046                          ValCysSerLeuCysSerCysThrAlaTrpProIleLeuGlyLeuPro                              110115120                                                                     CCCACCTGGTACAAGAGCTTCGAATACCGTGCGCGCGTGGTCCGCGAA1094                          ProThrTrpTyrLysSerPheGluTyrArgAlaArgValValArgGlu                              125130135140                                                                  CCACGGAAGGTTCTCTCCGAGATGGGAACCGAGATCGCGTCGGACATC1142                          ProArgLysValLeuSerGluMetGlyThrGluIleAlaSerAspIle                              145150155                                                                     GAGATTCGCGTCTACGACACCACCGCCGAAACTCGCTACATGGTCCTC1190                          GluIleArgValTyrAspThrThrAlaGluThrArgTyrMetValLeu                              160165170                                                                     CCGCAGCGTCCCGCCGGCACCGAAGGCTGGAGCCAGGAACAACTGCAG1238                          ProGlnArgProAlaGlyThrGluGlyTrpSerGlnGluGlnLeuGln                              175180185                                                                     GAAATCGTCACCAAGGACTGCCTGATCGGGGTTGCAATCCCGCAGGTT1286                          GluIleValThrLysAspCysLeuIleGlyValAlaIleProGlnVal                              190195200                                                                     CCCACCGTCTGATCACCCCGACAAGAAGGAAGCACACCATGGATGGAGTACAC1339                     ProThrValMetAspGlyValHis                                                      20515                                                                         GATCTTGCCGGAGTACAAGGCTTCGGCAAAGTCCCGCATACCGTCAAC1387                          AspLeuAlaGlyValGlnGlyPheGlyLysValProHisThrValAsn                              101520                                                                        GCCGACATCGGCCCCACCTTTCACGCCGAATGGGAACACCTGCCCTAC1435                          AlaAspIleGlyProThrPheHisAlaGluTrpGluHisLeuProTyr                              253035                                                                        AGCCTGATGTTCGCCGGTGTCGCCGAACTCGGGGCCTTCAGCGTCGAC1483                          SerLeuMetPheAlaGlyValAlaGluLeuGlyAlaPheSerValAsp                              404550                                                                        GAAGTGCGATACGTCGTCGAGCGGATGGAGCCGGGCCACTACATGATG1531                          GluValArgTyrValValGluArgMetGluProGlyHisTyrMetMet                              556065                                                                        ACCCCGTACTACGAGAGGTACGTCATCGGTGTCGCGACATTGATGGTC1579                          ThrProTyrTyrGluArgTyrValIleGlyValAlaThrLeuMetVal                              70758085                                                                      GAAAAGGGAATCCTGACGCAGGACGAACTCGAAAGCCTTGCGGGGGGA1627                          GluLysGlyIleLeuThrGlnAspGluLeuGluSerLeuAlaGlyGly                              9095100                                                                       CCGTTCCCACTGTCACGGCCCAGCGAATCCGAAGGGCGGCCGGCACCC1675                          ProPheProLeuSerArgProSerGluSerGluGlyArgProAlaPro                              105110115                                                                     GTCGAGACGACCACCTTCGAAGTCGGGCAGCGAGTACGCGTACGCGAC1723                          ValGluThrThrThrPheGluValGlyGlnArgValArgValArgAsp                              120125130                                                                     GAGTACGTTCCGGGGCATATTCGAATGCCTGCATACTGCCGTGGACGA1771                          GluTyrValProGlyHisIleArgMetProAlaTyrCysArgGlyArg                              135140145                                                                     GTGGGAACCATCTCTCATCGAACTACCGAGAAGTGGCCGTTTCCCGAC1819                          ValGlyThrIleSerHisArgThrThrGluLysTrpProPheProAsp                              150155160165                                                                  GCAATCGGCCACGGGCGCAACGACGCCGGCGAAGAACCGACGTACCAC1867                          AlaIleGlyHisGlyArgAsnAspAlaGlyGluGluProThrTyrHis                              170175180                                                                     GTGAAGTTCGCCGCCGAGGAATTGTTCGGTAGCGACACCGACGGTGGA1915                          ValLysPheAlaAlaGluGluLeuPheGlySerAspThrAspGlyGly                              185190195                                                                     AGCGTCGTTGTCGACCTCTTCGAGGGTTACCTCGAGCCTGCGGCC1960                             SerValValValAspLeuPheGluGlyTyrLeuGluProAlaAla                                 200205210                                                                     TGATCTTCCAGCATTCCAGGCGGCGGTCACGCGATCACAGCGGTTCGTGCGACCGCCGCC2020              TGATCACCACGATTCACTCATTCGGAAGGACACTGGAAATCATGGTCGAC2070                        (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2456 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Pseudomonas chlororaphis                                        (B) STRAIN: B23 (FERM BP-187)                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1021..1620                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1666..2325                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       GGATCCCGTCGGCCTTCTGCGGTACCTACGGCATGAAGCCCACCCACGGCCTGGTGCCCT60                ACACCGGCGTCATGGCGATTGAAGCCACGATCGATCATGTCGGCCCCATCACCGGTAACG120               TGCGCGACAACGCGCTGATGCTGCAGGCAATGGCCGGTGCAGACGGACTCGACCCGCGCC180               AGGCGGCGCCTCAGGTCGATGACTATTGCAGTTACCTGGAAAAAGGCGTGAGCGGACTCA240               GAATCGGGGTGTTGCAAGAGGGATTCGCGCTTGCTAACCAGGACCCTCGCGTGGCGGACA300               AAGTGCGCGACGCCATCGCCCGACTCGAGGCGTTGGGCGCTCATGTCGAGCCGGTCTCCA360               TTCCCGAGCACAACCTGGCAGGGTTGTTGTGGCACCCCATCGGTTGCGAAGGCTTGACCA420               TGCAGATGATGCATGGCAACGGCGCAGGCTTTAACTGGAAAGGACTTTACGATGTCGGCC480               TGCTGGACAAACAAGCCAGCTGGCGCGACGACGCAGACCAATTATCCGCGTCGCTCAAGC540               TCTGCATGTTCGTCGGCCAATACGGCCTGTCGCGCTACAACGGACGCTACTACGCCAAGG600               CCCAGAACCTTGCACGCTTTGCCCGGCAGGGATACGACAAAGCGCTGCAAACCTATGACC660               TGCTGGTGATGCCGACCACGCCCATCACGGCCCAACCCCACCCGCCAGCGAACTGCTCGA720               TCACGGAGTACGTGGCTCGCGCGTTGGAAATGATCGGCAATACCGCGCCACAGGACATCA780               CCGGGCATCCGGCCATGTCGATTCCGTGTGGCCTGCTGGACGGCCTGCCCGTCGGGCTGA840               TGCTGGTCGCAAAACACTACGCCGAGGGCACGATTTACCAAGCGGCGGCGGCGTTTGAAG900               CCTCGGTGGACTGGCGCACGCTCTGAGCCTTTTACAGGCGCCGCCCCCCCTGAAGAACGA960               TAAGAAAGACCGGCAAGTTGCAATGACTTTTCAACCGCGTTGACTGATAGGAGTTACCCC1020              ATGAGTACATCTATTTCCACGACTGCGACACCTTCGACACCCGGCGAG1068                          MetSerThrSerIleSerThrThrAlaThrProSerThrProGlyGlu                              151015                                                                        AGGGCATGGGCCTTGTTTCAAGTGCTCAAGAGCAAGGAACTCATCCCA1116                          ArgAlaTrpAlaLeuPheGlnValLeuLysSerLysGluLeuIlePro                              202530                                                                        GAGGGCTATGTCGAGCAGCTCACTCAATTGATGGCCCATGACTGGAGC1164                          GluGlyTyrValGluGlnLeuThrGlnLeuMetAlaHisAspTrpSer                              354045                                                                        CCGGAGAACGGCGCTCGCGTGGTCGCCAAGGCATGGGTCGATCCGCAG1212                          ProGluAsnGlyAlaArgValValAlaLysAlaTrpValAspProGln                              505560                                                                        TTCCGGGCGCTGCTGCTCAAGGACGGAACAGCCGCTTGCGCGCAGTTC1260                          PheArgAlaLeuLeuLeuLysAspGlyThrAlaAlaCysAlaGlnPhe                              65707580                                                                      GGCTACACCGGCCCACAAGGCGAATACATCGTCGCCCTGGAAGATACA1308                          GlyTyrThrGlyProGlnGlyGluTyrIleValAlaLeuGluAspThr                              859095                                                                        CCGGGGGTGAAGAACGTCATCGTCTGCAGCCTGTGCTCCTGCACCAAC1356                          ProGlyValLysAsnValIleValCysSerLeuCysSerCysThrAsn                              100105110                                                                     TGGCCGGTCCTCGGCCTGCCGCCCGAGTGGTACAAGGGCTTTGAGTTT1404                          TrpProValLeuGlyLeuProProGluTrpTyrLysGlyPheGluPhe                              115120125                                                                     CGTGCGCGCCTGGTCCGGGAGGGGCGCACCGTACTGCGCGAGCTGGGG1452                          ArgAlaArgLeuValArgGluGlyArgThrValLeuArgGluLeuGly                              130135140                                                                     ACGGAGTTGCCGAGCGACACGGTCATCAAAGTCTGGGATACCAGCGCC1500                          ThrGluLeuProSerAspThrValIleLysValTrpAspThrSerAla                              145150155160                                                                  GAAAGCCGTTACCTGGTGTTGCCGCAAAGGCCTGAAGGCTCTGAGCAC1548                          GluSerArgTyrLeuValLeuProGlnArgProGluGlySerGluHis                              165170175                                                                     ATGAGTGAAGAACAGCTTCAACAGCTGGTGACCAAAGACGTGCTGATT1596                          MetSerGluGluGlnLeuGlnGlnLeuValThrLysAspValLeuIle                              180185190                                                                     GGCGTCGCCCTGCCACGCGTTGGCTGAGAAAAAACAACTCATCATCGTTCAACT1650                    GlyValAlaLeuProArgValGly                                                      195200                                                                        TGCGGAGTTTTCATTATGGATGGCTTTCACGATCTCGGCGGTTTCCAAGGC1701                       MetAspGlyPheHisAspLeuGlyGlyPheGlnGly                                          1510                                                                          TTTGGCAAAGTGCCGCACACCATCAACAGCCTCAGCTACAAACAGGTT1749                          PheGlyLysValProHisThrIleAsnSerLeuSerTyrLysGlnVal                              152025                                                                        TTCAAGCAGGACTGGGAACACCTGGCCTATAGCTTGATGTTTGTCGGC1797                          PheLysGlnAspTrpGluHisLeuAlaTyrSerLeuMetPheValGly                              303540                                                                        GTTGACCAATTGAAAAAGTTCAGCGTGGACGAAGTGCGTCATGCCGTC1845                          ValAspGlnLeuLysLysPheSerValAspGluValArgHisAlaVal                              45505560                                                                      GAACGCCTGGACGTTCGCCAGCATGTCGGCACCCAGTACTACGAACGC1893                          GluArgLeuAspValArgGlnHisValGlyThrGlnTyrTyrGluArg                              657075                                                                        TACATCATCGCGACCGCCACGCTGCTGGTGGAAACGGGCGTTATCACC1941                          TyrIleIleAlaThrAlaThrLeuLeuValGluThrGlyValIleThr                              808590                                                                        CAGGCGGAGCTCGATCAGGCATTGGGTTCCCACTTCAAGCTGGCGAAC1989                          GlnAlaGluLeuAspGlnAlaLeuGlySerHisPheLysLeuAlaAsn                              95100105                                                                      CCCGCCCATGCGACAGGTCGCCCGGCGATCACCGGCAGGCCGCCCTTC2037                          ProAlaHisAlaThrGlyArgProAlaIleThrGlyArgProProPhe                              110115120                                                                     GAAGTGGGCGATCGGGTTGTGGTTCGAGACGAATATGTGGCGGGGCAT2085                          GluValGlyAspArgValValValArgAspGluTyrValAlaGlyHis                              125130135140                                                                  ATCCGCATGCCGGCCTACGTGCGCGGTAAGGAAGGCGTGGTCCTGCAC2133                          IleArgMetProAlaTyrValArgGlyLysGluGlyValValLeuHis                              145150155                                                                     CGCACCTCAGAGCAGTGGCCCTTCCCCGACGCCATTGGCCACGGCGAC2181                          ArgThrSerGluGlnTrpProPheProAspAlaIleGlyHisGlyAsp                              160165170                                                                     TTGAGCGCAGCCCATCAGCCTACCTACCACGTCGAGTTTCGCGTGAAA2229                          LeuSerAlaAlaHisGlnProThrTyrHisValGluPheArgValLys                              175180185                                                                     GATCTATGGGGTGACGCGGCAGATGACGGTTACGTCGTGGTCGATCTT2277                          AspLeuTrpGlyAspAlaAlaAspAspGlyTyrValValValAspLeu                              190195200                                                                     TTCGAAAGCTACTTGGATAAGGCCCCCGGTGCCCAAGCGGTGAACGCA2325                          PheGluSerTyrLeuAspLysAlaProGlyAlaGlnAlaValAsnAla                              205210215220                                                                  TGATTGAAGGCGCCCAGGCGGGCCGACTGCCGGTGACGGTCCTTTCCGGCTTCCTCGGCG2385              CCGGCAAAACCACCCTGCTCAACGCTATCCTGCGAAATCGCCAAGGACTGCGGGTCGCGG2445              TCATCGTCAAC2456                                                               __________________________________________________________________________

What is claimed is:
 1. A method of producing nitrile hydratase whichcomprises culturing a prokaryotic transformed cell containing arecombinant vector which is plasmid pPCN4 obtained from E. coliJM105/pPCN4 having Accession Number FERM BP-2779 and recovering nitrilehydratase from the culture.
 2. The method of producing nitrile hydrataseaccording to claim 1, which comprises culturing a transformed cell,wherein the transformed cell is an E. coli cell, and recovering nitrilehydratase from the culture.
 3. An isolated DNA fragment obtainable fromplasmid pPCN4 contained in E. coli JM105/pPCN4 having Accession NumberFERM BP-12779, said DNA fragment consisting of a 6.2 kb SphI-SalIfragment of plasmid pPCN4.